This proposal is based upon the hypothesis that the trophoblast has the capacity to regulate maternal immune responses during pregnancy, independent of the hormonal status of the mother or the site of implantation. A system for transplanting equine invasive trophoblast cells to ectopic sites has been developed as a means to assess the maternal immune responses to the developing conceptus. Preliminary studies demonstrate that transplanted trophoblast: 1) differentiate and function in sites other than the uterus, 2) evoke strong cellular and humoral immune responses by the recipient, 3) downregulate MHC class I antigens as they do after invading the uterus in normal pregnancy, and 4) survive for 28 days or longer after transplantation. Based on the trophoblast transplant model, this proposal would address the following questions: 1) Are the humoral and cellular immune responses to invasive trophoblast altered in a non-uterine mucosal site (vulvar mucosa) when compared to those which occur 1n normal pregnancy? Immune responses to trophoblast transplants would be compared to those induced by normal pregnancy or by skin allografting using the following assays: lymphocyte microcytotoxicity assay to determine the cytotoxic antibody titer, duration, and onset, Western blot technique to identify the molecular target(s) of the antibody response, ELISA capture assay to determine antibody isotypes, quantitative RT-PCR assay to identify cytokine profiles, and cytotoxic lymphocyte (CTL) assay to assess cellular immune functions. 2) What mechanisms do invasive trophoblast cells employ to regulate maternal immune responses? Molecular assays would be used to determine if invasive trophoblast cells control immune effector function by synthesizing immunosuppressive factors, such as indoleamine 2,3- dioxygenase (IDO) and FasL. Quantitative and in situ RT-PCR assays would be performed on allogeneic and MHC class I compatible trophoblast transplants to determine the presence and temporal expression of these transcripts. The proposed study should provide important insights into the relative roles of the trophoblast, the uterine site, and the pregnant state in influencing fetal evasion of maternal immune responses.